The present invention relates to novel processes for preparing the (4S) enantiomer of 4-(3,4-dichlorophenyl)-3,4-dihydro-1(2H)-naphthalenone (hereinafter also referred to as xe2x80x9cchiral tetralonexe2x80x9d or xe2x80x9c(4S) tetralonexe2x80x9d) and, more specifically, relates to the stereoselective microbial reduction of racemic 4-(3,4-dichlorophenyl)-3,4-dihydro-1(2H)-naphthalenone (hereinafter also referred to as xe2x80x9cracemic tetralonexe2x80x9d) to chiral tetralone.
The chiral tetralone prepared by the processes of the present invention may be further reacted to prepare pure cis-(1S)(4S)-N-methyl-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-1-naphthaleneamine, commonly referred to as sertraline. Sertraline is well known to be useful, for example, as an antidepressant and anorectic agent, and in the treatment of chemical dependencies, anxiety-related disorders, premature ejaculation, cancer and post-myocardial infarction.
Methods are known in the art for preparing sertraline, such as, for example, those described in U.S. Pat. Nos. 4,536,518; 4,777,288; 4,839,104; 4,855,500; 4,940,731; 4,962,128; 5,082,970; 5,130,338; 5,196,607; 5,248,699; 5,442,116; 5,463,126; 5,466,880; 5,597,826; and 5,750,794; and, in the paper of W. M. Welch, Jr. et al., appearing in the Journal of Medicinal Chemistry, Vol. 27, No. 11, p. 1508 (1984).
Several of the aforementioned patents relate to the synthesis of mixtures of cis- and trans-isomers of racemic N-methyl-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-1-naphthaleneamine. As described therein, the cis- and trans-isomers, as well as their (S) and (R) enantiomers, may be separated by methods known to those skilled in the art including, for example, fractional crystallization or chromatography.
It is also known to select for the ultimately desired chirality earlier on in the synthesis of sertraline. For example, the aforementioned U.S. Pat. No. 5,750,794 discloses a process for preparing chiral tetralone by reacting racemic tetralone with an asymmetric ketone reducing agent to yield the corresponding cis- or trans-alcohols depending upon the chirality of the asymmetric reagent employed, and then separating the alcohols and oxidizing the (1S,4S) and/or (1R,4S) alcohols to (4S)-tetralone.
It is also known in the art that chiral compounds can be synthesized using microorganisms, such as, fungi, e.g., yeast. For example, the use of yeasts to reduce ketones to chiral alcohols is well known. However, as is appreciated by those of skill in the relevant art, the chemical and optical yields, e.g., particular enantiomers and amounts thereof, of such microbial reductions generally vary substantially depending on, for example, the particular microorganism chosen, as well as the substituents of the starting material.
U.S. Pat. No. 5,049,497 discloses a process for resolving a racemic derivative of bicyclo[4.2.0]octane by contacting the derivative with Baker""s Yeast under conditions sufficient to give a mixture of a ketone and an alcohol of high enantiomeric purity. As described therein, only one enantiomer of the subject racemic ketone is reduced to give an alcohol.
U.S. Pat. No. 5,580,764 discloses an asymmetric reduction process which uses an intact microorganism, or a broken cell preparation thereof, to convert a cyclic ketone to the corresponding chiral alcohol.
U.S. Pat. No. 5,618,707 discloses a process for the stereoselective reduction of ketone substrates by adding the substrates to a culture broth of either Zygosaccharomyces bailii ATCC (American Type Culture Collection) No. 38924 or Schizosaccharomyces octosporus ATCC No. 2479, incubating the resultant mixture, and isolating the hydroxy compound through conventional means such as, for example, extraction with organic solvents, adsorption to resins, or chromatography for subsequent use as an intermediate in the preparation of a serum cholesterol lowering agent. The isolated hydroxy compound described therein was analyzed by chiral high performance liquid chromatography (HPLC), reverse-phase HPLC, or both. Consistent with what would be understood by one of skill in the relevant art, as described therein, many of the large number of microorganisms which were investigated for their ability to reduce the ketone group of the selected substrate failed to reduce the ketone group with the desired specificity or productivity.
It has now been unexpectedly found that a range of microorganisms, including fungi, e.g., yeasts, and actinomycetes, substantially stereoselectively reduce a racemic tetralone. More specifically, the subject stereoselective microbial reduction selectively reduces the (4R) tetralone of the racemic mixture while leaving the (4S) tetralone substantially unreacted. Moreover, the unwanted (4R) tetralol produced by the subject process can be oxidized and then racemized to racemic tetralone and the subject process repeated to yield even more (4S) tetralone. The (4S) tetralone produced by the subject process can be used in the synthesis of sertraline.
All of the documents cited herein, including the foregoing, are incorporated by reference herein in their entireties.
The present invention relates to microbiological reduction of carbonyl groups which comprises contacting a ketone compound, the racemic tetralone of formula (I), with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resultant mixture under suitable conditions such that a compound having a hydroxy group, specifically, the (4R) tetralol of formula (II) can be formed and accumulated in the medium, and a compound having the desired stereochemistry, the (4S) tetralone of formula (V) below, remains substantially unreacted. The (4S) tetralone of formula (V) below, i.e., chiral tetralone, can then be isolated by any suitable method, e.g., chromatography or crystallization. In addition, the (4R) tetralol of formula (II) can be separated from the compounds of formulae (III)-(V), oxidized and racemized to racemic tetralone and the subject stereoselective microbial reduction repeated to result in even more of the desired chiral ketone.
Accordingly, the present invention provides processes for carrying out the following stereospecific microbial reduction: 
which comprises: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resulting mixture under conditions sufficient to yield more of the compound of formula (II) than the compound of formula (III), thus leaving more of the compound of formula (V) unreacted than the compound of formula (IV) unreacted.
The subject stereospecific reduction may also be represented by: 
which comprises: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resulting mixture under conditions sufficient to yield the (4R) tetralol of formula (II) and to leave substantially unreacted the (4S) tetralone of formula (V).
The stereoselective reduction further optionally comprises the separation of the (4S) tetralone of formula (V) from the (4R) tetralol of formula (II). The (4R) tetralol may then be oxidized to produce the (4R) tetralone, which is then reacted, e.g., with a base, to produce racemic tetralone of formula (I) and the subject stereoselective microbial reduction may be repeated to result in even more of the desired (4S) tetralone of formula (V), i.e., the (4S) enantiomer of the racemic tetralone of formula (I).
The present invention provides processes which comprise the stereoselective microbial reduction of a compound of formula (I) to a compound of formula (II) by: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resulting mixture under conditions sufficient to yield a compound of formula (II), whereby substantially more of the compound of formula (V) remains unreacted than the compound of formula (IV) and substantially more of the compound of formula (II) is produced than the compound of formula (III).
In a preferred embodiment the contacting of the compound of formula (I) is with an enzyme reduction system. In another preferred embodiment the contacting of the compound of formula (I) is with an enzyme reduction system wherein the enzyme is immobilized. In a particularly preferred embodiment the contacting of the compound of formula (I) is with an enzyme reduction system derived from Hansenula polymorpha ATCC No. 26012.
In another preferred embodiment the microorganism is a broken cell preparation thereof. In yet another preferred embodiment the microorganism is an acetone powder enzymatic preparation thereof.
In an especially preferred embodiment of the present invention an intact microorganism is used. In a preferred embodiment wherein the microorganism is an intact microorganism, the compound of formula (I) is contacted with a fermentation medium, culture broth, or solvent, comprising the microorganism. In another preferred embodiment wherein the microorganism is an intact microorganism, the compound of formula (I) is contacted with washed intact microorganism. In yet another preferred embodiment wherein the microorganism is intact, the compound of formula (I) is contacted with immobilized intact microorganism.
In an especially preferred embodiment of the present invention the microorganism is an intact microorganism which is grown in a fermentation medium and the contacting occurs by the addition of the compound of formula (I) thereto.
In another especially preferred embodiment of the present invention the microorganism is an intact microorganism which is grown in a growth medium for about forty-eight hours, and the contacting occurs in such growth medium by the addition of the compound of formula (I) thereto, and the incubation is for about five days.
In another preferred embodiment of the present invention the microorganism is either a fungus, e.g., a yeast, or an actinomycete, or a mutant thereof which is capable of performing the stereoselective reduction.
In yet another preferred embodiment of the present invention the microorganism is a fungus. In still another preferred embodiment wherein the microorganism is a fungus, the fungus is Absidia coerulea ATCC No. 20137.
In a particularly preferred embodiment of the present invention the microorganism is a yeast. In an especially preferred embodiment of the present invention wherein the microorganism is a yeast, the yeast is Hansenula polymorpha ATCC No. 26012, also deposited as ATCC No. 74449.
Where Hansenula polymorpha ATCC No. 26012, also deposited as ATCC No. 74449, is employed as the microorganism, the subject stereoselective microbial reduction appreciably reduces only one enantiomer of the compound of formula (I), to give the corresponding alcohol, i.e., the compound of formula (II), while leaving the other enantiomer of the compound of formula (I), i.e., the compound of formula (V), substantially unreacted.
As discussed earlier, the processes of the present invention further optionally include the separation, e.g., carried out using crystallization or chromatography, of the compound of formula (V) from the compounds of formulae (II)-(IV), and the use of such separated compound of formula (V) in the synthesis of sertraline using any known methods therefor.
As also discussed earlier, it is preferred to oxidize the isolated (4R) tetralol of formula (II) to the (4R) tetralone of formula (IV). It is then further preferred to racemize, preferably by reacting the (4R) tetralone with a base, the (4R) tetralone of formula (IV) to the racemic tetralone of formula (I). The oxidation and racemization recycles the unwanted (4R) tetralol for another round of stereoselective microbial reduction according to the processes of the present invention. The recycling of the unwanted (4R) tetralol increases the amount of the desired (4S) tetralone and decreases the amount of unwanted (4R) tetralol discarded. The oxidation and the racemization of the oxidized product may be carried out using any suitable known methods therefor.
Those skilled in the art will fully understand the terms used herein to describe the present invention; nonetheless, the following terms used herein, are as described immediately below.
xe2x80x9cCo-factorxe2x80x9d means any suitable co-factor comprising the enzyme reduction system such as, for example, NADH, NADPH, FADH, FMNH, and/or PQQ or any suitable co-factor which occurs with the enzyme in the microorganism. xe2x80x9cEnzyme reduction systemxe2x80x9d means a suitable microbial oxidoreductase enzyme and the reduced form of a co-factor for the oxidoreductase enzyme, where the co-factor may either be derived from the selected microorganism or may be from any suitable source. The enzyme comprising the enzyme reduction system may be in either free or immobilized form, e.g., in a column or attached to a bead.
xe2x80x9cMicrobial reductionxe2x80x9d means the stereoselective reduction of the present invention as accomplished by the enzyme reduction system, the microbial reductase comprising the enzyme reduction system, the intact microorganism, or any preparation thereof, and the like.
xe2x80x9cMicroorganismxe2x80x9d includes any intact microorganism or preparation therefrom, including, for example, a broken cell preparation of the microorganism; a dehydrated preparation of the microorganism, e.g., an acetone powder enzymatic preparation; microorganism washed free of, e.g., fermentation medium, culture broth, and the like; microorganism immobilized, e.g., in a column, attached to beads, and the like.
The processes provided by the present invention comprise the stereoselective microbial reduction of a compound of the formula (I) to a compound of the formula (II): 
by contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resulting mixture under conditions sufficient to yield a compound of formula (II), whereby substantially more of the compound of formula (V) remains unreduced than the compound of formula (IV) and substantially more of the compound of formula (II) is produced than the compound of formula (III).
As would be understood by those skilled in the art, the compound of formula (I), racemic tetralone, is a mixture of (4S) tetralone and (4R) tetralone as shown below: 
The compounds, or more specifically, the tetralols of formula (II) are: 
The compounds, or more specifically, the tetralols of formula (III) are: 
The compounds of formulae (II) and (III) are disclosed and claimed in the aforementioned U.S. Pat. No. 5,750,794.
The desired compound of formula (V) may be isolated as described below from the undesired compounds of formula (II), and any of the compounds of formulas (III) or (IV) which may have been either produced or remained unreacted, respectively, depending upon, e.g., the microorganism selected and the conditions of incubation.
The compounds of formula (II) may be converted to a compound of formula (I), e.g., by oxidization and racemization, and run through the subject stereoselective microbial reduction to result in yet another amount of the (4S) tetralone of formula (V).
The process of the present invention is readily carried out. Thus, the microorganism is either fermented (intact microorganism) or incubated (broken cell preparation, dehydrated preparation, or any other suitable preparation of the microorganism) in the presence of the racemic tetralone, represented by formula (I), to modify racemic tetralone, and more particularly, to reduce the undesired (4R) enantiomer of the racemic ketone to its corresponding alcohol, represented by formula (II), while leaving the desired (4S) enantiomer, represented by formula (V), substantially unreacted, thereby, in one step, resulting in the optically enriched (4S) enantiomer. The (4S) enantiomer may then be further reacted by methods well known to those skilled in the relevant art such as described, for example, in the aforementioned U.S. Pat. Nos. 4,536,518; 4,777,288; 4,839,104; 4,855,500; 4,940,731; 4,962,128; 5,082,970; 5,130,338; 5,196,607; 5,248,699; 5,442,116; 5,463,126; 5,466,880; 5,597,826; and 5,750,794; and, in the aforementioned paper of W. M. Welch, Jr. et al., to ultimately yield sertraline.
The activity, methods for testing activities, dosages, dosage forms, methods of administration and background information concerning sertraline are set forth, for example, in the aforementioned U.S. Pat. Nos. 4,536,518; 4,777,288; and 4,839,104; and the aforementioned paper by W. M. Welch, Jr. et al.
Any suitable microorganism may be used in the process of the present invention. As described earlier, the microorganism used in the subject process may be intact, any suitable preparation thereof, e.g., a broken cell preparation thereof, a dehydrated preparation thereof, and be either free or immobilized. However, where a non-intact microorganism is employed in the present invention such as, for example, a broken cell preparation, e.g., cell extract, acetone powder enzymatic preparation, or the enzyme derived therefrom, those skilled in the art would understand that a suitable co-factor for the enzyme is also included.
Those skilled in the art will understand from the description provided herein and their related knowledge how to prepare a suitable broken cell preparation such as described, for example, by R. N. Patel et al. in the article xe2x80x9cOxidation of Secondary Alcohols to Methyl Ketones by Yeastsxe2x80x9d published in Applied and Environmental Microbiology, 38(2): 219-223 (1979).
Those skilled in the art will understand from the description provided herein and their related knowledge how to prepare a suitable acetone powder enzymatic preparation such as described, for example, by K. Nakamura et al. in the article xe2x80x9cAsymmetric Reduction of Ketones by the Acetone Powder of Geotrichum candidumxe2x80x9d published in Tetrahedron Letters, 37(10): 1629-1632 (1996).
In addition, an enzyme (e.g., an oxidoreductase) of any suitable microorganism may also be used in the subject processes, and this enzyme may be isolated from the microorganism by any suitable method known to those skilled in the art and, as for the intact microorganism, may be used in the subject process in either free or immobilized form. Those skilled in the art will understand from the description provided herein and their related knowledge how to isolate and purify the enzyme of the suitable microorganism such as generally described, for example, in the articles by: M. Wada et al., xe2x80x9cPurification and Characterization of NADPH-Dependent Carbonyl Reductase, Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate, from Candida magnoliaexe2x80x9d published in Biosci. Biotechnol. Biochem, 62(2): 280-285 (1998), P. Trost et al., xe2x80x9cPurification and Properties of NAP(P)H:(quinone-acceptor) oxidoreductase of sugarbeet cellsxe2x80x9d published in Eur. J. Biochem., 234: 452-458 (1995), K. M. Madyastha and T. L. Gururaja, xe2x80x9cPurification and Some of the Properties of a Novel Secondary Alcohol Dehydrogenase from Alcaligenes eutrophusxe2x80x9d published in Biochemical and Biophysical Research Communications, 211(2): 540-546 (1995), O. Bortolini et al., xe2x80x9cKinetic resolution of vic-diols by Bacillus stearothermophilus diacetyl reductasexe2x80x9d published in Tetrahedron: Asymmetry, 9: 647-651 (1998), R. N. Patel et al., xe2x80x9cStereospecific microbial reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(triflurormethyl-1H-1)-benzazepin-2-onexe2x80x9d published in Enzyme Microb. Technol., 3: 906-912 (1991) and R. N. Patel et al., xe2x80x9cStereoselective microbial/enzymatic oxidation of (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding chiral lactol and lactonexe2x80x9d published in Enzyme Microb. Technol., 14: 778-784 (1992); and by U.S. Pat. No. 5,523,223 and the aforementioned U.S. Pat. No. 5,580,764.
Suitable microorganisms include Hansenula polymorpha ATCC No. 26012, Hansenula polymorpha ATCC No. 74449, Absidia coerulea ATCC No. 20137, Geotrichum candidum ATCC No. 34614, Geotrichum candidum ATCC No. 62401, Mortierella isabellina ATCC No. 42613, Mortierella isabellina ATCC No. 38063, Mortierella vinacea ATCC No. 09515, Penicillium notatum ATCC No. 36740, Blastoschizomyces capitatus ATCC No. 28575, Monosporium olivaceum v. major ATCC No. 36300, Aureobasidium pullulans ATCC No. 16623, Debaryomyces polymorphus ATCC No. 20280, Saccharomyces cerevisiae ATCC No. 15248, Candida schatavii ATCC No. 24409, Pichia fabianii ATCC 16755 and Streptomyces rimosus ss. rimosus ATCC No. 10970; and mutants thereof which are known or otherwise obtainable by those skilled in the relevant art and able, despite such mutation, to accomplish the stereoselective microbial reduction disclosed herein.
Preferred intact microorganisms will be those which substantially reduce the (4R) tetralone while leaving the (4S) tetralone substantially unreacted where reacted includes reduction or any other intrinsic activity which might degrade or otherwise negatively impact the desired (4S) tetralone at any stage of the subject process. As would be appreciated by those skilled in the art from the disclosure herein, such unwanted reaction of the (4S) tetralone may be substantially prevented by, for example, using the enzyme derived from the selected microorganism versus the intact microorganism.
The microorganisms suitable for use in the subject stereospecific microbial reduction may be prepared by any suitable method known to those skilled in the relevant art. An example of a suitable method for the preparation of a microorganism from a commercially purchased stock is provided below. The method provided below may be used for any microorganism suitable for use in the present inventive process, and those skilled in the art would understand from the description provided herein how to modify any part of the procedure, e.g., method of preparing the microorganism, intact or preparation, e.g., broken cell or dehydrated, thereof, free or immobilized; method of preparing a suitable enzyme derived from such microorganisms; method of contacting of the racemic tetralone with the microorganism or the enzyme comprising the enzyme reduction system derived therefrom; growth medium components and conditions, e.g., temperature, pH and the like; or incubation conditions; to achieve the desired result in any particular process.
Those skilled in the art will understand from the description provided herein and their related knowledge how to prepare suitable immobilized intact microorganism such as described, for example, by A. Bauer et al. in the article xe2x80x9cPolyvinyl alcohol-immobilized whole-cell preparations for biotransformation of nitritesxe2x80x9d published in Biotechnology Letters, 18(3): 343-348 (March 1996).
Any suitable method of contacting the compound of formula (I) with the microorganism or enzyme reduction system may be used in the present invention. The compound of formula (I) may be contacted with the microorganism or the enzyme reduction system in any suitable order. For example, the compound of formula (I) may be added to a medium, such as a culture broth, comprising the microorganism, free or immobilized, or some combination thereof; or the medium may comprise the compound of formula (I) and the microorganism may then be added to such medium; or the compound of formula (I) and the microorganism may be added together to such medium; or the compound of formula (I) may be added to a broken cell preparation thereof; or the compound of formula (I) may be added to a dehydrated preparation of the microorganism; or either the compound of formula (I) or the microorganism or enzyme reduction system may be added to a suitable solvent comprising the other; and the like. Those skilled in the art will understand from the description provided herein how to modify any part of the subject process as so desired.
It is especially preferred in the present invention that the microorganism, or the enzyme reduction system, is derived from Hansenula polymorpha ATCC No. 26012. A lyophilized sample of Hansenula polymorpha ATCC No. 26012 (originally contributed by D. W. Levine) was deposited with the ATCC located at 10801 University Boulevard, Manassas, Va., 20110-2209, U.S.A., under the terms of the Budapest Treaty on Jun. 26, 1998. This newly deposited culture was given the new deposit number of ATCC No. 74449. Hence, it is also especially preferred in the present invention that the microorganism is Hansenula polymorpha ATCC No. 74449. All restrictions on the availability to the public of the microorganism culture so deposited will be irrevocably removed upon the issuance of a patent from the specification of the present invention.
Cultures of Hansenula polymorpha ATCC No. 26012 can be obtained from the ATCC, and an example of a suitable method for its preparation from such a commercially purchased stock is provided immediately below. A culture so obtained is added to a suitable growth medium, and is incubated with shaking until growth occurs, both steps as would be appreciated by those skilled in the art. The culture, thus prepared, can be used to inoculate slants, with portions of these slants frozen as master stocks. Alternatively, liquid stock cultures can be prepared to which glycerol is added to from about 10% to about 20% which are then frozen at about xe2x88x9280xc2x0 C., preferably in small cryotubes.
As would be understood by those skilled in the art for any microorganism selected, and as provided specifically hereinafter in the examples for Absidia coerulea ATCC No. 20137 and the especially preferred Hansenula polymorpha ATCC No. 26012 or ATCC No. 74449, a suitable method for preparing the microorganism is as follows: the microorganism is inoculated from a frozen stock culture such as described above (about a 17% glycerol stock) into a flask or a glass tube with a metal closure containing a growth medium (containing an aliquot from a sterile solution which includes Tween(copyright) 80, glycerol and distilled water) whose composition is described in more detail below. The fermentation is carried out at temperatures ranging from about 22xc2x0 C. to about 32xc2x0 C., and preferably at about 29xc2x0 C., with suitable shaking, preferably from about 200 rpm to about 220 rpm, and most preferably, at about 210 rpm. Where so desired, the pH of the growth medium can be maintained by the use of suitable buffers incorporated into the fermentation medium and/or periodically adjusted by addition of either base or acid as so required.
Any suitable duration of growth of the microorganism, contacting of the microorganism with the compound of formula (I), and incubation of the compound of formula (I) with the microorganism may be used in the present invention. Suitable growth of the microorganism may be achieved, e.g., within about 24 hours, at which time a suitable aliquot of a solution of racemic tetralone in a suitable solvent, preferably ethanol, may be added to the culture. The fermentation may then be continued for, e.g., from about two to about six days, and preferably, e.g., for about five days, at which time the fermentation broth may be extracted using any suitable extraction method whereby a suitable solvent, such as, for example, ethyl acetate, methyl isobutylketone, methyl ethylketone, methylene chloride, and the like, preferably, ethyl acetate, removes the organic components from the fermentation broth. After extraction of the fermentation broth and separation of the organic and aqueous phases, the compounds comprising the organic residue may be determined using any suitable method, such as, for example, chromatography, preferably, chiral HPLC.
Any suitable growth medium may be used in the process of the present invention, and the suitable growth medium will contain a source or sources of assimilable carbon, assimilable nitrogen and inorganic salts containing essential minerals. In general, many carbohydrates such as, for example, glucose, maltose, mannose, sucrose, starch, glycerin, millet jelly, molasses, soy bean, and the like, can be used as sources of assimilable carbon. Sources of assimilable nitrogen include, for example, materials such as yeast and casein hydrolysates, primary yeast, yeast extracts, cottonseed flour, soybean solids, wheat germ, meat extracts, peptone, corn steep liquor, and ammonium salts. Suitable inorganic salt nutrients for use in the culture medium of the present invention include, for example, the customary salts containing sodium, iron, magnesium, potassium, cobalt, phosphate, and the like. More particularly, growth media suitable for use in the present invention include, for example:
(a) dextrose (about 20 gram (g)), yeast extract (about 5 g), soy flour (about 5 g), NaCl (about 5 g), K2HPO4 (about 5 g), and distilled H2O (about 1 liter (L)), pH adjusted to about pH 7.0 with H2SO4(aq.);
(b) dextrin (about 10 g), beef extract (about 3 g), ardamine pH (about 5 g), NZ amine type E (about 5 g), MgSO47H2O (about 0.5 g), KH2PO4 (about 0.37 g), CaCO3 (about 0.5 g), and distilled H2O (about 1 L), pH adjusted to about pH 7.1 with HCl(aq.), followed by a second stage of glucose (about 10 g), Hy-Case SF(copyright) (about 2 g), beef extract (about 1 g), corn steep liquor (about 3 g), and distilled H2O (about 1 L), pH adjusted to about pH 7.0;
(c) glucose (about 10 g), corn steep liquor (about 6 g), KH2PO4 (about 3 g), CaCO3 (about 3.5 g), soybean oil (crude, about 2.2 milliliters (ml)), yeast extract (about 2.5 g), and distilled H2O (about 1 L), pH adjusted to from about pH 7.0 to about pH 7.3 with HCl(aq.);
(d) malt syrup (about 20 g), soybean meal (about 5 g), casein (about 1 g), dried yeast (about 1 g), NaCl (about 5 g), and distilled H2O (about 1 L);
(e) lactose (about 75 g), Pharmamediae(copyright) (substitute yeast extract, about 40 g), CaCO3 (about 10 g), Na2SO4 (about 4 g), and distilled H2O (about 1 L);
(f) ISP #2 (see, e.g., page 460 of the Handbook of Microbial Media by R. M. Atlas, edited by L. C. Parks, CRC Press, Inc., 1993, (xe2x80x9cHandbookxe2x80x9d));
(g) ISP #3 (see, page 460 of the Handbook);
(h) ISP#4 (see, page 461 of the Handbook);
(i) ISP#5 (see, pages 461-462 of the Handbook); and the like.
A particularly preferred growth medium is 2xc3x97 of (a) provided above.
Reference to particular buffers, media, reagents, contacting or culture conditions, and the like, is not intended to be limiting, but should be read to include all such related materials that those of ordinary skill in the art would recognize as being of interest or value in the particular context in which the discussion herein is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. Moreover, it should be understood that the present invention includes the scaling-up of the subject process for commercial purposes.
Hence, as would be understood by those of ordinary skill in the art, variation of the growth medium, the conditions of fermentation, and/or the amount of racemic tetralone may be altered to control the yield of the resultant compounds and their relative rates of production. In general, the techniques employed in the present invention will be chosen with regard to industrial efficiency. The growth media, conditions of fermentation and relative amounts of microorganism, or enzyme reduction system, and racemic tetralone described herein are merely illustrative of the wide variety of media, fermentation conditions and amounts of starting materials which may be suitably employed in the present invention as would be appreciated by those skilled in the art, and are not intended to be limiting in any way.
Any suitable methods for isolating and/or purifying any of the products of the subject process may be used in the present invention including filtration, extraction, crystallization, column chromatography, thin-layer chromatography, preparative low pressure liquid chromatography or HPLC, or any suitable combination of such methods.
Further, one of skill in the art would appreciate that the unwanted corresponding alcohol of the (4R) tetralone, the compound of formula (II), produced by the processes disclosed herein, may be recycled, e.g., oxidized and racemized as discussed earlier herein, by any suitable known method to a racemic tetralone of formula (I), and the processes of the present invention repeated to result in, once again, the desired (4S) tetralone of formula (V). The oxidation of the (4R) tetralol to the (4R) ketone can be done by methods known to those skilled in the art. The racemization reaction may be performed in any suitable manner but is generally performed at a temperature of from about 0xc2x0 C. to about 100xc2x0 C., preferably from about 25xc2x0 C. to about 65xc2x0 C. The (4R) tetralone is reacted with a base at a temperature of from about 25xc2x0 C. to about 85xc2x0 C., preferably from about 50xc2x0 C. to about 65xc2x0 C. Suitable bases for this racemization reaction include potassium t-butoxide, sodium hydroxide, sodium methoxide and potassium hydroxide. A preferred base is potassium t-butoxide.
The detailed examples provided below show that a range of microorganisms, including fungi, e.g., yeasts, and actinomycetes, stereoselectively reduce racemic tetralone, to yield the desired (4S) tetralone of formula (V), i.e., chiral tetralone, which may then be separated from the unwanted compounds and further reacted according to methods well known in the art to yield sertraline.
The present invention is illustrated by the following examples. The foregoing and following description of the present invention and the various embodiments are not intended to be limiting of the invention but rather are illustrative thereof. Hence, it will be understood that the invention is not limited to the specific details of these examples.